Method for detecting biological material

ABSTRACT

The present invention provides a staining method in which the fluorescent staining properties in a fluorescently-immunostained specimen are not reduced even when an oil-based mounting medium is used. The present invention also provides a method of preventing deterioration of a fluorescent label caused by irradiation with excitation light and improving the light resistance in a fluorescently-immunostained specimen obtained by the staining method. The biological substance detection method according to the present invention is a biological substance detection method for specifically detecting a biological substance from a pathological specimen, which includes the steps of: immunostaining the specimen with a fluorescent label; immobilizing the thus stained specimen; and mounting the thus immobilized specimen using a mounting medium including an organic solvent not freely miscible with water. In the biological substance detection method, the above-described mounting medium further includes a discoloration inhibitor.

TECHNICAL FIELD

The present invention relates to a biological substance detectionmethod. More particularly, the present invention relates to tissuestaining in which a tissue is stained with a fluorescent label.

BACKGROUND ART

As a medical diagnosis, pathological diagnosis is performed. Apathologist diagnoses a disease using a tissue section collected fromhuman body and informs a clinician of whether or not a therapy and/orsurgery is/are necessary. Based on the patient conditions and thepathological diagnosis, a physician determines pharmacotherapeuticstrategies and a surgeon determines whether or not a surgery should beperformed.

In pathological diagnosis, it is a common practice to prepare a tissuespecimen by slicing a tissue sample obtained by evisceration or needlebiopsy into a thickness of about several micrometers and then observethe tissue specimen at a magnification under a light microscope so as toobtain various findings. In many cases, a specimen is prepared by fixinga collected tissue through dehydration and paraffin blocking, slicingthe thus fixed tissue into a thickness of several micrometers, and thenremoving the paraffin.

In pathological diagnosis, immunological observation in which moleculartarget staining called immunostaining is performed for verifying theexpression of molecular information of a specimen and functionalabnormalities such as abnormal expression of a gene or a protein arediagnosed is performed. For immunostaining, for example, a dye stainingmethod using an enzyme (e.g., DAB staining) is employed. In DABstaining, an antibody modified with peroxidase, which is capable ofallowing diaminobenzidine (DAB) to show a color, is used to stain anantigen to be observed with the color and the amount of the antigen isdetermined by observing the stained antigen. Alternatively, fluorescentlabeling may be employed in some cases. In fluorescent labeling, theamount of the subject antigen is determined by staining the antigen withan antibody modified with a fluorescent dye and observing the stainedantigen.

Further, since a specimen hardly absorbs or scatters any light and isthus nearly colorless and transparent, it may be subjected to stainingwith a die for morphological observation prior to being observed. Therehave been proposed a variety of staining techniques. In particular, fortissue specimens, hematoxylin-eosin staining (HE staining) using twodyes, hematoxylin and eosin, is typically used as staining for observingthe morphology of the subject specimen (Non-patent Document 1, PatentDocuments 1 and 2). Hematoxylin stains cell nuclei, calcareous parts,cartilaginous tissues, bacteria and mucus in livid to light blue, whileeosin stains cytoplasm, interstitial tissues, various fibers,erythrocytes and keratinocyte in red to dark red. A pathologist makes adiagnosis based on the morphological and staining information, such aschanges in the size and shape of cell nuclei and changes in the patternas a tissue, in a micrograph of the stained tissue specimen. Examples ofother staining for morphological observation include Papanicolaoustaining (Pap staining) used for cytological diagnosis. By subjecting atissue section to both morphological staining and immunostaining,morphological observation and immunological observation of the specimencan be performed simultaneously.

At present, DAB staining is often used in immunological observation(Patent Document 3). However, in staining with an enzyme label such asDAB staining, since the staining concentration is largely variabledepending on the environmental conditions such as temperature and time,there is a problem that estimation of the actual amount of an antibodyor the like from the staining concentration is difficult. Therefore, forimmunological observation in pathological diagnosis, fluorescentlabeling using a fluorescent label is also performed as an alternativeto staining with an enzyme label. This method characteristically hassuperior quantitative capability than DAB staining (Non-patent Document1).

Observation using a fluorescent label is performed under a confocallaser scanning microscope or epifluorescence microscope. Suchmicroscopes utilize a high-intensity excitation light. For example, asopposed to an intensity of 1,000 W/m², which is normal sunlight exposuretest condition described in the solar cell standard JIS C 8914, theintensity of the irradiation light used by a typical epifluorescencemicroscope is 100 times stronger.

When the fluorescent label is damaged by excitation light and no longeremits light, a reduction in the signal occurs. Therefore, in theobservation using a fluorescent label, the light resistance of thefluorescent label is important.

It is known that fluorescent dyes, inorganic nanoparticles (which mayalso be referred to as “semiconductor nanoparticle”, “quantum dot” orthe like) and aggregates thereof are utilized as fluorescent labels(Non-patent Documents 2 and 3, Patent Document 4). It has been reportedthat, as compared to fluorescent dyes and inorganic nanoparticles,aggregates thereof have an improved light resistance (Patent Document5). Therefore, from the standpoint of the light resistance, suchaggregates are more preferred; however, the aggregates alone do not havesufficient light resistance required for fluorescence microscopy. Here,an aggregate has a higher brightness than that of one dye molecule;therefore, from the standpoint of the signal, an aggregate is morepreferred.

In preparation of a specimen, aqueous mounting media and oil-basedmounting media are known as mounting media for mounting a stainedpathological section. Aqueous mounting media have a problem in that,since their refractive indices are largely different from that of aspecimen, it is difficult to make a specimen transparent and to producea permanent preparation thereof. Meanwhile, oil-based mounting media arecharacterized in that their refractive indices are not largely differentfrom that of a specimen and can thus make the specimen transparent; andthat they are typically used for preparing a permanent preparation thatshows good color tone and color development in morphological staining.Accordingly, oil-based mounting media are more preferably used in thepreparation of a specimen.

Therefore, also as a mounting medium for an immunostained specimen, apermanent preparation can be produced with an oil-based mounting mediumand, when a specimen is subjected to double staining of immunostainingand morphological staining, it is believed that the use of an oil-basedmounting medium showing good color tone and color development inmorphological staining is more preferred.

However, when a fluorescently-stained specimen is mounted using anoil-based mounting medium, there is a problem that the fluorescent dyeelutes into the mounting medium and this impairs the stainingproperties. Therefore, there is a circumstance that, while an oil-basedmounting medium can be used when the specimen is stained without usingany fluorescent label, an aqueous mounting medium having theabove-described problems must be used when the specimen is stained witha fluorescent label.

In addition, for observation of a fluorescent label, a confocal lasermicroscope or a fluorescence microscope is used. In fluorescenceobservation under these microscopes, a stained section is irradiatedwith a high-intensity excitation light. This excitation light graduallydeteriorates a fluorescent label containing a fluorescent dye or thelike and this has a great effect in the fluorescence observation andassessment of immunostaining results. Ideally, there is no deteriorationof such fluorescent label. Otherwise, it is necessary to improve thelight resistance of the fluorescent label.

In order to improve the light resistance of a fluorescent label, it isthought to admix a discoloration inhibitor with a mounting medium. Inaqueous mounting media, for example, incases where4′,6-diamidino-2-phenylindole (DAPI) is used as a fluorescent dye, it isattempted to improve the light resistance of DAPI by using adiscoloration inhibitor such as 1,4-diazabicyclo[2.2.2]octane (DABCO) orProLong Gold (registered trademark, manufactured by Molecular ProbesInc.). However, although the use of such discoloration inhibitoralleviates the deterioration of the fluorescent label, since it cannotcompletely inhibit the deterioration, observation must be made in ashort period.

Nevertheless, since the use of an oil-based mounting medium in stainingwith a fluorescent dye has the above-described problems, oil-basedmounting media have not been used in staining with a fluorescent dye.Therefore, there is no motivation to admix a discoloration inhibitor toan oil-based mounting medium and to use the oil-based mounting medium influorescent staining, and such an attempt has thus not been made.

PRIOR ART REFERENCES Patent Documents

[Patent Document 1] Japanese Translated PCT Patent Application Laid-openNo. 2001-525580

[Patent Document 2] Japanese Laid-open Patent Application (Kokai) No.2009-115599

[Patent Document 3] Japanese Laid-open Patent Application (Kokai) No.2010-134195

[Patent Document 4] Japanese Laid-open Patent Application (Kokai) No.2010-209314

[Patent Document 5] Japanese Laid-open Patent Application (Kokai) No.2008-147394

Non-Patent Documents

[Non-patent Document 1] “Shindan ni yakudatsu men-eki soshiki Shindan(Immunohistochemistry Useful for Diagnosis)”, Bunkado Co., Ltd., 2007

[Non-patent Document 2] “Synthesis and Applied Technology of FunctionalDyes”, CMC Publishing Co., Ltd., 2007

[Non-patent Document 3] “Application of Quantum Dot in Life Science”,CMC Publishing Co., Ltd., 2007

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a staining method inwhich the fluorescent staining properties in afluorescently-immunostained specimen are not reduced even when anoil-based mounting medium is used. Another object of the presentinvention is to provide a method of preventing deterioration of afluorescent label caused by irradiation with excitation light andimproving the light resistance in a fluorescently-immunostained specimenobtained by the staining method. Yet another object of the presentinvention is to provide a kit used for these methods.

Means for Solving the Problems

The present inventors intensively studied to discover that, in stainingwith a fluorescent label, by immobilizing the subject specimen afterbinding the fluorescent label thereto, the fluorescent stainingproperties are not reduced even when an oil-based mounting medium isused to mount the specimen; and that, by incorporating a discolorationinhibitor into the oil-based mounting medium used in this process, thelight resistance of the fluorescent label can be largely improved,thereby completing the present invention. In one aspect of the presentinvention, in order to realize at least one of the above-describedobjects, the present invention includes the following items.

[1] A biological substance detection method for specifically detecting abiological substance from a pathological specimen, the method comprisingthe steps of: immunostaining the specimen with a fluorescent label;immobilizing the thus stained specimen; and mounting the thusimmobilized specimen using a mounting medium comprising an organicsolvent not freely miscible with water.

[2] The biological substance detection method according to [1], whereinthe above-described mounting medium comprises a discoloration inhibitor.

[3] A biological substance detection kit used for the biologicalsubstance detection method according to [1], the kit comprising: amounting medium comprising an organic solvent not freely miscible withwater; and an instruction manual that describes the above-describedbiological substance detection method.

[4] A biological substance detection kit used for the biologicalsubstance detection method according to [2], the kit comprising: amounting medium comprising an organic solvent not freely miscible withwater and a discoloration inhibitor; and an instruction manual thatdescribes the above-described biological substance detection method.

[5] A pathological specimen used in the biological substance detectionmethod according to [1], wherein the pathological specimen is subjectedto: an immunostaining treatment using a fluorescent label forspecifically detecting a biological substance; an immobilizationtreatment; and amounting treatment using a mounting medium comprising anorganic solvent not freely miscible with water.

Effects of the Invention

According to the biological substance detection method of [1], aspecimen in which the fluorescent staining properties are not impairedin immunostaining with a fluorescent label can be prepared and apermanent preparation can also be prepared. Further, when doublestaining of immunostaining and morphological staining is performed, aspecimen showing good color tone and color development in morphologicalstaining can be prepared.

According to the biological substance detection method of [2], inaddition to the above-described effects, a specimen in which the lightresistance of a fluorescent label is improved can be prepared as well.

By using the biological substance detection kit according to [3], aspecimen prepared by the method of [1] can be obtained.

By using the biological substance detection kit according to [4], aspecimen prepared by the method of [2] can be obtained.

The pathological specimen according to [5] is a specimen or permanentpreparation obtained by the method of [1] in which the fluorescentstaining properties are not impaired, and it is a pathological specimenwhich, when subjected to double staining of immunostaining with afluorescent label and morphological staining, shows good color tone andcolor development in morphological staining.

MODE FOR CARRYING OUT THE INVENTION

The mode for carrying out the invention will now be described; however,the present invention is not restricted thereto.

The biological substance detection method according to a typicalembodiment of the present invention is a method of specificallydetecting a biological substance from a pathological specimen, whichbasically comprises: (1) the step of immunostaining a pathologicalspecimen with a fluorescent label; and (2) the step of irradiating thethus stained pathological specimen with excitation light to allowfluorescence to be emitted for detection of a biological substance fromthe pathological specimen. In the present invention, the step (1)comprises the specimen immobilization step and mounting step.

The step (1) may also comprise other steps such as deparaffinizationstep and activation step in the same manner as in a general biologicalsubstance detection method.

Further, either before or after the step (1), the step of staining apathological specimen with a staining agent for morphologicalobservation may be incorporated as well. By this, immunostaining with afluorescent label and morphological staining with a staining agent formorphological observation can be performed simultaneously.

In the present invention, particularly in the step (1) of immunostaininga pathological specimen, (A) a fluorescent dye, (B) a fluorescentnanoparticle, (C) a fluorescent dye-containing nanoparticle or (D) afluorescent nanoparticle-containing particle can be used as thefluorescent label. From the standpoint of the signal value ratio tonoises, which are the fluorescence of eosin and the intrinsicfluorescence of cells, the higher the brightness of the fluorescentlabel, the more preferred it is. Accordingly, in the present invention,(B) a fluorescent nanoparticle, (C) a fluorescent dye-containingnanoparticle and (D) a fluorescent nanoparticle-containing particle,which have a higher brightness than (A) a fluorescent dye, can besuitably used as the fluorescent label. Further, from the standpoint ofattaining higher light resistance, an aggregate of (C) fluorescentdye-containing nanoparticles and an aggregate of (D) fluorescentnanoparticle-containing particles can be particularly suitably used.

The details of the fluorescent label for immunostaining, theimmunostaining step, the staining agent for morphological observationand the morphological staining step will now be described below.

[Fluorescent Label for Immunostaining]

In the present invention, the fluorescent label for immunostaining maybe any existing fluorescent label as long as it can be used forimmunostaining. The fluorescent label for immunostaining may be any of(A) a fluorescent dye, (B) a fluorescent nanoparticle, (C) a fluorescentdye-containing nanoparticle and (D) a fluorescentnanoparticle-containing particle.

[(A) Fluorescent Dye]

The fluorescent dye used in the present invention may be any existingfluorescent dye. It can be obtained or prepared by a known method.

The fluorescent dye to be contained can be selected from, for example,rhodamine-based dye molecules, squarylium-based dye molecules,cyanine-based dye molecules, aromatic ring-based dye molecules,oxazine-based dye molecules, carbopyronine-based dye molecules andpyrromethene-based dye molecules. Alternatively, the fluorescent dye tobe contained can also be selected from, for example, Alexa Fluor(registered trademark, manufactured by Invitrogen)-based dye molecules,BODIPY (registered trademark, manufactured by Invitrogen)-based dyemolecules, Cy (registered trademark, manufactured by GEHealthcare)-based dye molecules, DY (registered trademark, DyomicsGmbH)-based dye molecules, HiLyte (registered trademark, manufactured byAnaSpec Inc.)-based dye molecules, DyLight (registered trademark,manufactured by Thermo Fisher Scientific K.K.)-based dye molecules, ATTO(registered trademark, manufactured by ATTO-TEC GmbH)-based dyemolecules and MFP (registered trademark, manufactured by Mobitec Co.,Ltd.)-based dye molecules. The generic names of these dye molecules aredesignated based on the main structure (skeleton) or registeredtrademark of the respective compounds; therefore, those of ordinaryskill in the art can properly understand the scope of the fluorescentdyes belonging to the respective generic names without having to bearundue trial and error.

Specific examples of the rhodamine-based dye molecules include5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine,rhodamine 6G, tetramethyl rhodamine, X-rhodamine, Texas Red, SpectrumRed and LD700 PERCHLORATE.

Specific examples of the squarylium-based dye molecules include SRfluor680-carboxylate,1,3-bis[4-(dimethylamino)-2-hydroxyphenyl]-2,4-dihydroxycyclobutenediylium dihydroxide,bis,1,3-bis[4-(dimethylamino)phenyl]-2,4-dihydroxycyclobutene diyliumdihydroxide,bis,2-(4-(diethylamino)-2-hydroxyphenyl)-4-(4-(diethyliminio)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1-enolate,2-(4-(dibutylamino)-2-hydroxyphenyl)-4-(4-(dibutyliminio)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1-enolate,and2-(8-hydroxy-1,1,7,7-tetramethyl-1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinolin-9-yl)-4-(8-hydroxy-1,1,7,7-tetramethyl-2,3,6,7-tetrahydro-1H-pyrido[3,2,1-ij]quinolinium-9(5H)-ylidene)-3-oxocyclobut-1-enolate.

Specific examples of the cyanine-based dye molecules include1-butyl-2-[5-(1-butyl-1,3-dihydro-3,3-dimethyl-2H-indol-2-ylidene)-penta-1,3-dienyl]-3,3-dimethyl-3H-indoliumhexafluorophosphate,1-butyl-2-[5-(1-butyl-3,3-dimethyl-1,3-dihydro-indol-2-ylidene)-3-chloro-penta-1,3-dienyl]-3,3-dimethyl-3H-indoliumhexafluorophosphate, and3-ethyl-2-[5-(3-ethyl-3H-benzothiazol-2-ylidene)-penta-1,3-dienyl]-benzothiazol-3-iumiodide.

Specific examples of the aromatic ring-based dye molecules includeN,N-bis-(2,6-diisopropylphenyl)-1,6,7,12-(4-tert-butylphenoxy)-perylene-3,4,9,10-tetracarbonaciddiimide,N,N′-bis(2,6-diisopropylphenyl)-1,6,7,12-tetraphenoxyperylene-3,4:9,10-tetracarboxdiimide,N,N′-bis(2,6-diisopropylphenyl)perylene-3,4,9,10-bis(dicarbimide),16,N,N′-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylicdiimide,4,4′-[(8,16-dihydro-8,16-dioxodibenzo[a,j]perylene-2,10-diyl)dioxy]dibutyricacid, 2,10-dihydroxy-dibenzo[a,j]perylene-8,16-dione,2,10-bis(3-aminopropoxy)dibenzo[a,j]perylene-8,16-dione,3,3′-[(8,16-dihydro-8,16-dioxodibenzo[a,j]perylene-2,10-diyl)dioxy]dipropylamine,17-bis(octyloxy)anthra[9,1,2-cde-]benzo[rst]pentaphene-5-10-dione,octadecanoic acid,5,10-dihydro-5,10-dioxoanthra[9,1,2-cde]benzo[rst]pentaphene-16,17-diylester,dihydroxydibenzanthrone, benzenesulfonic acid,4,4′,4″,4′″-[[2,9-bis[2,6-bis(1-methylethyl)phenyl]-1,2,3,8,9,10-hexahydro-1,3,8,10-tetraoxoanthra[2,1,9-def:6,5,10-d′e′f′]diisoquinoline-5,6,12,13-tetrayl]tetrakis(oxy)]tetrakis,benzeneethanaminium, and4,4′,4″,4′″-[[2,9-bis[2,6-bis(1-methylethyl)phenyl]-1,2,3,8,9,10-hexahydro-1,3,8,10-tetraoxoanthra[2,1,9-def:6,5,10-d′e′f′]diisoquinoline-5,6,12,13-tetrayl]tetrakis(oxy)]tetrakis[N,N,N-trimethyl-].

Specific examples of the oxazine-based dye molecules include Cresylviolet, Oxazine 170, EVOblue 30 and Nile Blue.

Specific examples of the carbopyronine-based dye molecules includeCARBOPYRONIN 149.

Specific examples of the pyrromethene-based dye molecules includePYRROMETHENE 650.

Specific examples of the Alexa Fluor-based dye molecules include AlexaFluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, AlexaFluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, AlexaFluor 680, Alexa Fluor 700 and Alexa Fluor 750 (all of which aremanufactured by Invitrogen).

Specific examples of the BODIPY-based dye molecules include BODIPY FL,BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650 and BODIPY650/665 (all of which are manufactured by Invitrogen).

Specific examples of the Cy-based dye molecules include Cy 3.5, Cy 5 andCy 5.5 (all of which are manufactured by GE Healthcare).

Specific examples of the DY-based dye molecules include DY-590, DY-610,DY-615, DY-630, DY-631, DY-632, DY-633 and DY-634 (all of which aremanufactured by Dyomics GmbH).

Specific examples of the HiLyte-based dye molecules include HiLyte 594and HiLyteFluor TR (both of which are manufactured by AnaSpec Inc).

Specific examples of the DyLight-based dye molecules include DyLight 594and DyLight 633 (both of which are manufactured by manufactured byThermo Scientific).

Specific examples of the ATTO-based dye molecules include ATTO 590, ATTO610, ATTO 620, ATTO 633 and ATTO 655 (all of which are manufactured byATTO-TEC GmbH).

Specific examples of the MFP-based dye molecules include MFP 590 and MFP631 (both of which are manufactured by Mobitec Co., Ltd.).

Examples of other dyes include C-phycocyanin, phycocyanin, APC(allophycocyanin), APC-XL and NorthernLights 637 (all of which aremanufactured by R&D Systems, Inc.).

Further, examples of other dyes also include derivatives of theabove-described dyes (which can function as a fluorescent dye, such asknown derivatives).

[(B) Fluorescent Nanoparticle]

The fluorescent nanoparticle used in the present invention has aparticle size of 1 to 500 nm, preferably 10 to 200 nm.

The fluorescent nanoparticle is composed of a semiconductor orfluorophore.

As the semiconductor, for example, a group II-VI semiconductor such asZnSe, ZnTe, CdSe, CdTe, PbS, PbSe or PbTe, or a group II-VIsemiconductor such as AlAs, AlSb, GaP, GaAs, GaSb, InP, InAs or InSb canbe used. From the standpoint of toxicity, GaP or InP can be suitablyused.

In the fluorophore, for example, Y₂O₃, YVO₄, ZnO or ZnS can be used asthe matrix and Eu or Nd can be used as the emission center.

An excitation wavelength suitable for observation is attained byadjusting the particle size, matrix composition and impurity amount ofthe fluorescent nanoparticle.

[(C) Fluorescent Dye-Containing Nanoparticle]

From the standpoint of the signal value ratio to noises, which are thefluorescence of eosin and the intrinsic fluorescence of cells, thehigher the brightness of the fluorescent label, the more preferred itis. Accordingly, in the present invention, a fluorescent dye-containingnanoparticle having a higher brightness than a fluorescent dye issuitably used as the fluorescent label.

The term “fluorescent dye-containing nanoparticle” refers to anano-sized particle having a structure in which plural fluorescent dyesare contained in a particle (matrix) made of an organic or inorganicmaterial. The fluorescent dye-containing nanoparticle used in thepresent invention can be prepared by a known method upon selecting, asraw materials, appropriate fluorescent dyes and particle-forming organicor inorganic material.

Examples of the particle-forming organic or inorganic material includethose which are capable of stably containing fluorescent dyes, such aspolystyrene, polyamide, polylactic acid, polyacrylonitrile, polyglycidylmethacrylate, polymelamine, polyurea, polybenzoguanamine, polyfuran,polyxylene, phenol resins, polysaccharides and silica. When fluorescentdyes are incorporated into such a particle, deterioration caused byirradiation with excitation light is less likely to occur (higher lightresistance is attained) as compared to a case where the fluorescent dyesare used by themselves.

As the fluorescent dyes to be contained, those fluorescent dyes that areexemplified above for the (A) fluorescent dye can be used. In addition,for example, derivatives thereof (which can function as a fluorescentdye, such as known derivatives) can also be used.

In the fluorescent dye-containing nanoparticle, any one of theabove-described fluorescent dyes may be contained individually, or aplurality thereof may be contained in combination.

For example, fluorescent dyes such as aromatic ring-based dye moleculesand rhodamine-based dye molecules are preferred because of theirrelatively high light resistance. Thereamong, perylenes belonging to thearomatic ring-based dye molecules, particularly perylene diimide, ispreferred. Meanwhile, even when a fluorescent dye having a relativelylow light resistance is used, by selecting an appropriate matrix, it ispossible to produce a fluorescent dye-containing nanoparticle whichsatisfies the prescribed condition of brightness retention rateaccording to the present invention.

The method of producing the fluorescent dye-containing nanoparticle isnot particularly restricted. For introduction of a dye(s) into aparticle, any method such as a method of synthesizing a particle bybinding a dye molecule (s) to a monomer used as raw material of theparticle or a method of introducing a dye(s) to a particle by adsorptionmay be employed.

The average particle size of the fluorescent dye-containing nanoparticleis not particularly restricted; however, it is usually 10 to 500 nm,preferably 50 to 200 nm. Further, the variation coefficient whichindicates the variation in the particle size is also not particularlyrestricted; however, it is usually 20% or less, preferably 5 to 15%.Here, the particle size of a fluorescent dye-containing nanoparticle canbe determined by taking an electron micrograph thereof using a scanningelectron microscope (SEM), measuring the cross-sectional area of thefluorescent dye-containing nanoparticle and then determining theparticle size as the diameter of a circular area corresponding to themeasured value (area equivalent circle diameter). With regard to theaverage particle size (average particle diameter) and the variationcoefficient of a group of fluorescent dye-containing nanoparticles,after measuring the particle sizes (particle diameters) for a sufficientnumber (for example, 1,000) of fluorescent dye-containing nanoparticlesin the above-described manner, the average particle size is calculatedas the arithmetic mean of the measured values and the variationcoefficient is calculated by the following equation: 100×(standarddeviation of particle size)/(average particle size).

[(D) Fluorescent Nanoparticle-Containing Particle]

The fluorescent nanoparticle-containing particle used in the presentinvention is a particle made of an organic or inorganic material thatcontains the fluorescent nanoparticles explained in the above (B). Inthe fluorescent nanoparticle-containing particle, any one of theabove-described fluorescent nanoparticles may be contained individually,or a plurality thereof may be contained in combination.

The method of producing the fluorescent nanoparticle-containing particleis not particularly restricted. For introduction of a fluorescentnanoparticle(s) into a particle, any method such as a method ofsynthesizing a particle by binding a fluorescent nanoparticle(s) to amonomer used as raw material of the particle or a method of introducinga fluorescent nanoparticle(s) to a particle by adsorption may beemployed. An excitation wavelength suitable for observation is attainedby adjusting the particle size, matrix composition and impurity amountof the fluorescent nanoparticle(s) to be contained.

The fluorescent nanoparticle-containing particle has a size of usually10 to 500 nm, preferably 50 to 200 nm.

[Step of Staining for Morphological Observation]

In the present invention, staining can be performed for morphologicalobservation. In the step of staining for morphological observation,particularly when the morphology of a tissue specimen is observed, theabove-described hematoxylin-eosin staining (HE staining) which utilizestwo dyes (hematoxylin and eosin) is typically employed; however, in thepresent invention, the staining for morphological observation is notrestricted thereto. Examples of other staining for morphologicalobservation include Papanicolaou staining (Pap staining) used forcytological diagnosis.

In HE staining, hematoxylin stains cell nuclei, calcareous parts,cartilaginous tissues, bacteria and mucus in livid to light blue, whileeosin stains cytoplasm, interstitial tissues, various fibers,erythrocytes and keratinocyte in red to dark red. In other staining formorphological observation, a hematoxylin analogue or a dye having anabsorption wavelength similar to that of hematoxylin may stain cellnuclei, calcareous parts, cartilaginous tissues, bacteria and mucus inlivid to light blue, and an eosin analogue or a dye having absorptionand emission wavelengths similar to those of eosin may stain cytoplasm,interstitial tissues, various fibers, erythrocytes and keratinocyte inred to dark red.

[Immunostaining Step]

In the present invention, as an immunostaining method, a fluorescentstaining method in which a biological substance to be detected isstained with the above-described fluorescent label for immunostaining isemployed.

For example, when immunostaining a specific antigen, a method in which alabel (conjugate) is prepared by directly binding a fluorescent labeland a primary antibody and an antigen is then stained (primary antibodymethod), a method in which a label is prepared by directly binding afluorescent label and a secondary antibody and an antigen bound with aprimary antibody is then stained (secondary antibody method), or amethod in which a label is prepared by directly binding a fluorescentlabel and biotin and an antigen bound with a primary antibody and avidinor a streptavidin-modified secondary antibody is then stained(biotin-avidin method or sandwich method) can be employed.

Any primary antibody may be used in the immunostaining and the primaryantibody is variable depending on the subject to be immunostained. Forexample, when immunostaining is performed using HER2 as an antigen, ananti-HER2 antibody is used. Further, any secondary antibody may be usedand the secondary antibody is variable depending on the primaryantibody. Examples thereof include anti-mouse, rabbit, bovine, goat,sheep, dog and chicken antibodies.

For binding of a fluorescent label with an antibody or biotin, anyexisting method may be employed. For example, amidation by reactionbetween amine and carboxylic acid, sulfidation by reaction betweenmaleimide and thiol, imination by reaction between aldehyde and amine,or amination by reaction between epoxy and amine can be used.

Here, the above-described immunostaining is not restricted to tissuestaining and can be applied to cell staining as well. Further, thebiological substance to be detected is not particularly restricted aslong as a substance which specifically binds thereto is present.Typically, a combination of an antigen and an antibody is used asdescribed above; however, it is also possible to use, for example, acombination of a nucleic acid molecule (oligonucleotide orpolynucleotide) and a nucleic acid molecule having a sequence that canbe complementarily coupled thereto.

[Immobilization Step]

The immobilization step performed in the staining method of the presentinvention is a step of immobilizing the fluorescent label introduced bythe above-described immunostaining step onto a biological substance, anantibody bound thereto or the like. By performing a treatment with animmobilization solution, proteins are cross-linked and denatured, sothat the fluorescent label can be chemically and physically bound morefirmly in a stable state to the biological substance, the antibody boundthereto or the like. In the present invention, such immobilizationtreatment can be carried out by immersing the stained tissue sectionobtained by the histochemical staining step in an immobilizationsolution. For example, the stained tissue section obtained by thehistochemical staining step can be immersed in a dilute paraformaldehydeaqueous solution for several minutes to several hours or so.

Examples of an immobilization solution that can be used in the presentinvention include cross-linking agents and cell membrane permeablesubstances, such as formalin, paraformaldehyde, glutaraldehyde, acetone,ethanol and methanol. Thereamong, formalin, paraformaldehyde andglutaraldehyde can be preferably used because they are capable ofattaining strong immobilization.

[Mounting Step]

The mounting step comprises the dehydration and clearing step using anorganic solvent of the subject tissue section and the mounting stepusing an oil-based mounting medium. In the dehydration and clearingstep, after washing the stained tissue section with an aqueous washingsolution such as PBS, the tissue section is dehydrated with EtOH(ethanol) and then substituted with xylene. The dehydration with EtOH iscarried out by immersing the tissue section sequentially in EtOH whosewater content is reduced to, for example, 50%, 30%, 10% and 0% andthereby substituting the tissue section with EtOH. By immersing the thusEtOH-substituted tissue section in xylene, the tissue section issubstituted with xylene and cleared. By placing the thusxylene-substituted tissue section on an oil-based mounting medium andputting a cover glass or the like thereon, the tissue section ismounted.

[Mounting Medium]

A mounting medium is constituted by a solvent (mounting solvent) and aresin. In the present invention, as a mounting medium, an oil-basedmounting medium (may be generally referred to as “non-aqueous mountingmedium”) is used. The term “oil-based mounting medium” refers to amounting medium which comprises a solvent not freely miscible withwater.

Here, the phrase “not freely miscible with water” means that the solventhas a volume-based solubility of 15% or less in water.

Further, the mounting medium of the present invention may also contain adiscoloration inhibitor. Such a mounting medium is hereinafter referredto as “discoloration inhibitor-containing mounting medium”.

The mounting medium may be a commercially available oil-based mountingmedium or a mounting medium prepared uniquely. Examples of thecommercially available oil-based mounting medium include DPX(manufactured by Sigma-Aldrich; main components: polystyrenepolymer=about 21.8%, xylene=about 69.7%), Entellan New (registeredtrademark, manufactured by Merck KGaA; main components: acrylic resinand xylene=about 60%) and PARA Mount-N (registered trademark,manufactured by FALMA; main components: acrylic resin and aliphatichydrocarbons). Some of the commercially available oil-based mountingmedia can be used as is, while others can be used after diluting theproduct (stock solution) with a prescribed solvent. Alternatively, bydissolving a natural resin such as Canada balsam or a synthetic resinsuch as polystyrene or polymethyl methacrylate into a mounting solvent,an oil-based mounting medium can be prepared uniquely.

<Mounting Solvent>

In the present invention, as a mounting solvent, a solvent whichcomprises an organic solvent not freely miscible with water is used. Themounting solvent corresponds to, for example, a solvent contained in theabove-described commercially available oil-based mounting media, asolvent prescribed for diluting the commercially available oil-basedmounting media, or a solvent used for uniquely preparing an oil-basedmounting medium.

The organic solvent not freely miscible with water can be selected fromaromatic hydrocarbons, unsaturated hydrocarbons, carbonyl-containingcompounds (ketones), esters, ethers and alcohols.

Thereamong, as an aromatic hydrocarbon, for example, benzene, tolueneand xylene can be used. As an unsaturated hydrocarbon, for example,limonene and pinene can be used. As a ketone, for example, cyclohexanoneand methyl ethyl ketone can be used. As an ester-based organic solvent,for example, butyl acetate can be used. As an ether, for example,anisole, 1,4-di(2-hydroxyethoxy)benzene and ethylene glycol monophenylether can be used. As an alcohol, for example, butanol, pentanol andhexanol can be used. Particularly, xylene, toluene and limonene arepreferred because of their availability, refractive index of about 1.5,which is close to that of a tissue section, and drying rate of severalten minutes or so, which is operationally suitable.

Any one of the above-described organic solvents not freely miscible withwater may be used individually, or a plurality thereof may be used incombination.

Further, the mounting solvent may be constituted only by an organicsolvent not freely miscible with water or, as required, in addition toan organic solvent not freely miscible with water, the mounting solventmay also contain water and/or an organic solvent that is freely misciblewith water in such an amount that does not inhibit the actions andeffects of the present invention. In a mounting medium not freelymiscible with water, the ratio (vol %) of the solvent not freelymiscible with water that is contained in the mounting solvent is usually50 to 100%, preferably 70 to 100%, more preferably 90 to 100%. Such amounting medium that comprises a solvent not freely miscible with waterin the prescribed range is preferred not only because the differencebetween the refractive index thereof and that of a specimen is small, sothat the specimen can be made transparent and a permanent preparationshowing good color tone and color development in morphological stainingcan be easily prepared, but also because such a mounting medium cancreate a more dehydrated condition, so that contamination by waterduring the preparation of a specimen can be easily inhibited and thespecimen can thus be evenly dried.

<Resin>

Since the resin contained in the mounting medium remains as solid afterthe solvent component is evaporated, as the resin, one which does notadversely affect the observation is suitable. Accordingly, a resin whichis highly transparent having a refractive index close to that of glassis appropriate. Further, since coloration and fluorescence adverselyaffect fluorescence microscopy, it is preferred that the resin haveneither of them. The resin used in the present invention is notparticularly restricted as long as it satisfies the above-describedconditions, and examples thereof include natural resins such as Canadabalsam and synthetic resins such as polystyrene and polymethylmethacrylate.

[Discoloration Inhibitor]

As the discoloration inhibitor, one which does not have any problem interms of the solubility in the mounting solvent comprising a solvent notfreely miscible with water can be selected from phenolic, amine-based,phosphorus-based, sulfur-based and unsaturated hydrocarbon-baseddiscoloration inhibitors.

Examples of the phenolic discoloration inhibitors that can be usedinclude phenols that are derived from natural products, such as rutin,catechin, hesperidin, methyl hesperidin, myricitrin, quercetin, morin,fisetin, naringenin, naringin, hesperidin, taxifolin, apigenin, geosmin,luteolin, cyanidin, delphinidin, malvidin, pelargonidin, peonidin,tannin, tocopherol and tocotrienol; and hindered phenols such asp-phenylazophenol, 4-nitroaniline,2,6-di-tert-butyl-4-hydroxymethylphenol,N,N′-disalicylal-1,2-propanediamine, triethyleneglycol-bis[3-(3-t-butyl-5-methyl-4-hydroxyphenyl)propionate],1,6-hexanediol-bis[3-(3,5-di-t-butyl-4-hydroxyphenyl)propionate],2,4-bis-(n-octylthio)-6-(4-hydroxy-3,5-di-t-butylanilino)-1,3,5-triazine,pentaerythrityl-tetrakis[3-(3,5-di-t-butyl-4-hydroxyphenyl)propionate],2,2-thio-diethylene-bis[3-(3,5-di-t-butyl-4-hydroxyphenyl)propionate],octadecyl-3-(3,5-di-t-butyl-4-hydroxyphenyl)propionate,N,N′-hexamethylene-bis(3,5-di-t-butyl-4-hydroxy-hydrocinnamamide),3,5-di-t-butyl-4-hydroxybenzyl phosphonate diethyl ester,1,3,5-trimethyl-2,4,6-tris(3,5-di-t-butyl-4-hydroxybenzyl)benzene,tris(3,5-di-t-butyl-4-hydroxybenzyl)isocyanurate, octylated diphenylamine, 2,4,-bis[(octylthio)methyl]-o-cresol,isooctyl-3-(3,5-di-t-butyl-4-hydroxyphenyl)propionate and1,3,5-tris(4-t-butyl-3-hydroxy-2,6-dimethylbenzyl)isocyanurate.

Examples of the amine-based discoloration inhibitors that can be usedinclude tertiary amines such as 1,4-diazabicyclo[2.2.2]octane (DABCO);secondary amines such as phenothiazine,bis(2,2,6,6-tetramethyl-4-piperidyl)sebacate and2,2,6,6-tetramethylpiperidine; and alkaloids such as caffeine.

Examples of the phosphorus-based discoloration inhibitors that can beused include 2-mercaptobenzimidazole, triphenyl phosphite,tris(2-carboxyethyl)phosphine hydrochloride (TCEP HCl), diisodecylpentaerythritol diphosphite and9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide.

Examples of the sulfur-based discoloration inhibitors that can be usedinclude sulfides such as didodecyl-3,3′-thiodipropionate and2,2′-thiodiethanol; disulfides such as dibenzyl disulfide, DL-α-lipoicacid (thioctic acid) and 3,6-dithia-1,8-octanediol; and thiols such asdithiothreitol and octanediol.

Examples of the unsaturated hydrocarbon-based discoloration inhibitorsthat can be used include carotenes, carotenoids, xanthophylls, ascorbicacid, tocotrienol and unsaturated fatty acid, such as lutein, lycopene,astaxanthin, canthaxanthin, capsanthin, Myxoxanthophyll, zeaxanthin,carotene and retinoic acid.

It is preferred that the above-described discoloration inhibitorscomprise at least one selected from the group consisting of phenols andamines. Such discoloration inhibitors can prevent photobleaching moreeffectively by inhibiting the oxidation of the fluorescent label byactive oxygen that causes photobleaching.

Any one of the above-described discoloration inhibitors may be usedindividually, or a plurality thereof may be used in combination.

In order to prevent any adverse effect in fluorescence microscopy, it ispreferred that these discoloration inhibitors show no absorption at awavelength of 450 to 600 nm and do not emit light at a wavelength of 500to 700 nm. Absorption of light leads to a reduction in the brightness ofthe fluorescent label. Further, emission of light leads to an increasednoise during fluorescence microscopy. Here, the expression that adiscoloration “shows no absorption” means that, when a xylene solutioncontaining the discoloration inhibitor at a concentration of 1 mg/mL isprepared and placed in a 10-mm cell and the absorbance is measured, theabsorbance is 0.5 or less at both 450 nm and 600 nm.

In the present invention, from the standpoint of preventing a reductionin the brightness of fluorescent dye(s), the section slide preparedusing a mounting medium containing the above-described discolorationinhibitor(s) is preferably transparent. The term “transparent” usedherein means that the absorbance of the prepared section slide ismeasured to be 0.1 or less at both 450 nm and 600 nm.

[Fluorescence Observation Step]

By irradiating the pathological specimen subjected to immunostaining andmorphological staining in the above-described steps with an excitationlight having a wavelength appropriate for the fluorescent label in use,the fluorescence emitted by the fluorescent label is observed. By thisstep, a prescribed biomolecule existing in the pathological specimen canbe detected and this information can be utilized to determine, forexample, the appropriateness of applying an antibody pharmaceutical(e.g., Herceptin which targets HER2).

For the irradiation of excitation light, the same irradiation means asthe one used in an ordinary fluorescence observation may be employed.For example, from a laser light source installed in a fluorescencemicroscope, an excitation light having an appropriate wavelength andoutput may be irradiated to the stained pathological specimen using, asrequired, a filter which selectively allows light having a prescribedwavelength to pass therethrough.

Observation of fluorescence may be performed either through the lensbarrel of a fluorescence microscope or on a separate display means(e.g., a monitor) showing an image taken by a camera mounted on afluorescence microscope. Depending on the fluorescent substance, evenwhen the fluorescence cannot be adequately observed visually through thelens barrel of a fluorescence microscope, the fluorescence may beobserved on an image taken by a camera in some cases. As required, afilter which selectively allows light having a prescribed wavelength topass therethrough may also be used.

Here, in the present invention, there are cases where immunostaining andmorphological staining are both performed on the same pathologicalspecimen. When observing an image produced by the morphologicalstaining, it is not required to irradiate the pathological specimen withexcitation light for exciting the fluorescent label used in theimmunostaining and the image may be observed under the same observationconditions as those of a light microscope.

Therefore, the fluorescence observation can be performed afterirradiating excitation light for an arbitrary time; however, thefluorescence observation is performed preferably within 90 minutes afterinitiating the irradiation of excitation light, more preferably within30 minutes after initiating the irradiation of excitation light, undernormal irradiation conditions (e.g., irradiation energy).

It is desired that the brightness of the fluorescent label should notchange before and after the observation under a fluorescence microscope.In the present invention, under observation conditions where an ordinaryfluorescence microscope has its diaphragm fully opened and is used at amagnification of ×40, the fluorescent label retains 70% of thepre-irradiation brightness after being irradiated for 30 minutes. Withthe brightness reduction being in this range, the biological substancedetection method is deemed to have certain reliability and can thus beapplied to observation of fluorescent immunostaining.

[Kit]

The biological substance detection kit of the present invention is usedfor the biological substance detection method of the present inventionwhich comprises the above-described steps. The biological substancedetection kit of the present invention comprises the above-describedmounting medium containing a solvent not freely miscible with water, orthe above-described mounting medium containing a solvent not freelymiscible with water and a discoloration inhibitor. The biologicalsubstance detection kit of the present invention may further comprise:an instruction manual which describes the biological substance detectionmethod according to the present invention as an instruction; afluorescent label for immunostaining; and a fluorescent labelingreagent.

The fluorescent label for immunostaining to be included in the kit canbe selected from those fluorescent labels that are described in theabove section [Fluorescent Label for Immunostaining]. As the fluorescentlabeling reagent, any of those substances that are described in theabove section [Immunostaining Step], for example, a primary antibody ora secondary antibody, can be selected. The mounting medium is the sameas the one described in the above section [Mounting Medium], and thesolvent and resin that constitute the mounting medium can each beselected from those solvents and resins that are described in the abovesections [Mounting Solvent] and [Resin], respectively. The discolorationinhibitor can be selected from those discoloration inhibitors that aredescribed in the above section [Discoloration Inhibitor].

<Instruction Manual>

The instruction manual to be included in the kit is one which describesany one of the biological substance detection methods according to thepresent invention as an instruction for carrying out the methodaccording to the present invention. The instruction manual takes anyspecific embodiment as long as it can properly convey theabove-described information. For example, the instruction manual may beprinted on a piece of paper, the package of the kit, or the label of aconstituent of the kit. Alternatively, the instruction manual may berecorded on a computer-readable medium such as a CD.

EXAMPLES

The present invention will now be described in detail by way of examplesthereof; however, the present invention is not restricted to thefollowing examples.

<Preparation of Fluorescent Labels>

The fluorescent labels were all prepared in a streptavidin-bound form.

(Fluorescent Label 1: Texas Red Dye)

As a fluorescent dye, Sulforhodamine 101 acid chloride (Texas Red dye,manufactured by Dojinsha Co., Ltd.) was used. Streptavidin was bound tothe fluorescent dye as follows.

Texas Red dye was adjusted with PBS (phosphate-buffered physiologicalsaline) containing 2 mM of EDTA (ethylenediamine tetraacetic acid) to aconcentration of 3 nM. The resulting solution was mixed with SM(PEG)12(manufactured by Thermo Fisher Scientific K.K.;succinimidyl-[(N-maleimidopropionamido)-dodecaethylene glycol]ester) toa final concentration of 10 mM and allowed to react for 1 hour. The thusobtained mixture was centrifuged at 10,000 G for 20 minutes and theresulting supernatant was removed. Then, PBS containing 2 mM of EDTA wasadded to disperse the precipitates and the resulting dispersion wascentrifuged again. The precipitates were washed three times by the sameprocedure to obtain fluorescent dye-containing nanoparticles having amaleimide group at a terminal (The unit M represents molarconcentration, mol/L).

Meanwhile, streptavidin (manufactured by Wako Pure Chemical Industries,Ltd.) was subjected to a thiol group addition treatment withN-succinimidyl-5-acetylthioacetate (SATA) and then filtered through agel-filtration column to obtain a solution of streptavidin capable ofbinding to dye-containing nanoparticles.

The above-described Texas Red dye and streptavidin were mixed in PBScontaining 2 mM of EDTA and allowed to react for 1 hour. Then, thereaction was terminated with an addition of 10 mM mercaptoethanol. Afterconcentrating the resulting solution using a centrifugation filter,unreacted streptavidin and the like were removed using a gel-filtrationcolumn for purification, thereby obtaining a streptavidin-bound TexasRed dye.

(Fluorescent Label 2: Perylene Dye)

Perylene diimide, which was used as a fluorescent dye, was prepared bythe following method.N,N′-bis(2,6-diisopropylphenyl)-1,6,7,12-tetraphenoxyperylene-3,4:9,10-tetracarboxdiimidewas treated with concentrated sulfuric acid to prepare a perylenediimide sulfonic acid derivative. This was then converted to an acidchloride to obtain a perylene diimide sulfonic acid chloride derivative.A streptavidin-bound dye was obtained in the same manner as thefluorescent dye 1, except that the thus obtained perylene diimidesulfonic acid chloride derivative was used.

(Fluorescent Label 3: Texas Red Dye-Containing Melamine Nanoparticle)

After adding 2.5 mg Sulforhodamine 101 (manufactured by Sigma-Aldrich)to 22.5 mL of water, the resulting mixture was heated at 70° C. for 20minutes on a hot stirrer and 1.5 g of a melamine resin, Nikalac MX-035(manufactured by Nippon Carbide Industries Co., Ltd.), was addedthereto, followed by heating of the resultant with stirring for another5 minutes. Then, 100 μL of formic acid was further added and theresultant was heated with stirring at 60° C. for 20 minutes andsubsequently cooled to room temperature. Thereafter, the resultingreaction mixture was placed in a centrifugal tube and centrifuged at12,000 rpm for 20 minutes, followed by removal of the resultingsupernatant. The precipitates were washed with ethanol and water.

Then, 0.1 mg of the thus obtained particles were dispersed in 1.5 mL ofEtOH and 2 μL of aminopropyltrimethoxysilane, LS-3150 (manufactured byShin-Etsu Chemical Co., Ltd.), was added thereto. The resulting mixturewas allowed to react for 8 hours so as to perform surface aminationtreatment.

The thus obtained dye-containing nanoparticles were adjusted with PBS(phosphate-buffered physiological saline) containing 2 mM of EDTA(ethylenediamine tetraacetic acid) to a concentration of 3 nM. Theresulting solution was mixed with SM(PEG)12 (manufactured by ThermoFisher Scientific K.K.;succinimidyl-[(N-maleimidopropionamido)-dodecaethylene glycol]ester) toa final concentration of 10 mM and allowed to react for 1 hour. The thusobtained mixture was centrifuged at 10,000 G for 20 minutes and theresulting supernatant was removed. Then, PBS containing 2 mM of EDTA wasadded to disperse the precipitates and the resulting dispersion wascentrifuged again. The precipitates were washed three times by the sameprocedure to obtain fluorescent dye-containing nanoparticles having amaleimide group at a terminal.

Meanwhile, streptavidin (manufactured by Wako Pure Chemical Industries,Ltd.) was subjected to a thiol group addition treatment withN-succinimidyl-5-acetylthioacetate (SATA) and then filtered through agel-filtration column to obtain a solution of streptavidin capable ofbinding to dye-containing nanoparticles.

The above-described fluorescent nanoparticles and streptavidin weremixed in PBS containing 2 mM of EDTA and allowed to react for 1 hour.Then, the reaction was terminated with an addition of 10 mMmercaptoethanol. After concentrating the resulting solution using acentrifugation filter, unreacted streptavidin and the like were removedusing a gel-filtration column for purification, thereby obtainingmelamine nanoparticles containing streptavidin-bound Texas Red dye.

(Fluorescent Label 4: Perylene Dye-Containing Melamine Nanoparticle)

The perylene diimide sulfonic acid chloride derivative described aboveas the dye 2 was used as a fluorescent dye.

Except this, melamine nanoparticles containing streptavidin-boundperylene dye were obtained in the same manner as the above-describedfluorescent dye 3.

(Fluorescent Label 5: FITC Dye-Containing Melamine Nanoparticle)

Melamine nanoparticles containing streptavidin-bound FITC dye wereobtained in the same manner as the above-described fluorescent dye 3,except that fluorescein isothiocyanate (FITC, manufactured by DojinshaCo., Ltd.) was used as the fluorescent dye.

(Fluorescent Label 6: Texas Red Dye-Containing Silica Nanoparticle)

In DMF, 3.4 mg of the Texas Red dye used in the fluorescent label 1 and3 μL of 3-aminopropyltrimethoxysilane (KBM903, manufactured by Shin-EtsuChemical Co., Ltd.) were mixed to obtain an organoalkoxysilane compound.Subsequently, 0.6 mL of the thus obtained organoalkoxysilane compoundwas mixed for 3 hours with 48 mL of ethanol, 0.6 mL of TEOS(tetraethoxysilane), 2 mL of water and 2 mL of 28% aqueous ammonia. Themixture produced in the above-described step was centrifuged at 10,000 Gfor 20 minutes and the resulting supernatant was removed. Then, ethanolwas added to disperse the precipitates and the resulting dispersion wascentrifuged again. The precipitates were washed twice by the sameprocedure with ethanol and pure water, thereby obtaining Texas Reddye-containing silica nanoparticles.

The thus obtained dye-containing nanoparticles were adjusted with PBS(phosphate-buffered physiological saline) containing 2 mM of EDTA(ethylenediamine tetraacetic acid) to a concentration of 3 nM. Theresulting solution was mixed with SM(PEG)12 (manufactured by ThermoFisher Scientific K.K.;succinimidyl-[(N-maleimidopropionamido)-dodecaethylene glycol]ester) toa final concentration of 10 mM and allowed to react for 1 hour. The thusobtained mixture was centrifuged at 10,000 G for 20 minutes and theresulting supernatant was removed. Then, PBS containing 2 mM of EDTA wasadded to disperse the precipitates and the resulting dispersion wascentrifuged again. The precipitates were washed three times by the sameprocedure to obtain fluorescent dye-containing nanoparticles having amaleimide group at a terminal.

Meanwhile, streptavidin (manufactured by Wako Pure Chemical Industries,Ltd.) was subjected to a thiol group addition treatment withN-succinimidyl-5-acetylthioacetate (SATA) and then filtered through agel-filtration column to obtain a solution of streptavidin capable ofbinding to dye-containing nanoparticles.

The above-described fluorescent nanoparticles and streptavidin weremixed in PBS containing 2 mM of EDTA and allowed to react for 1 hour.Then, the reaction was terminated with an addition of 10 mMmercaptoethanol. After concentrating the resulting solution using acentrifugation filter, unreacted streptavidin and the like were removedusing a gel-filtration column for purification, thereby obtaining silicananoparticles containing streptavidin-bound Texas Red dye.

(Fluorescent Label 7: Perylene Dye-Containing Silica Nanoparticle)

Silica nanoparticles containing streptavidin-bound perylene dye wereobtained in the same manner as the above-described fluorescent dye 6,except that perylene diimide used in the fluorescent label 2 was used asthe fluorescent dye.

(Fluorescent Label 8: FITC Dye-Containing Silica Nanoparticle)

Silica nanoparticles containing streptavidin-bound perylene dye wereobtained in the same manner as the above-described fluorescent dye 6,except that FITC used in the fluorescent dye 3 was used as thefluorescent dye.

(Fluorescent Label 9: Texas Red Dye-Containing Polystyrene Nanoparticle)

Texas Red dye-containing polystyrene particles were prepared by asoap-free emulsion polymerization method. A fluorescent dye,Sulforhodamine 101 acid chloride (Texas Red dye, manufactured byDojinsha Co., Ltd.), was mixed with 4-aminostyrene (manufactured byTokyo Chemical Industry Co., Ltd.) at room temperature for 1 hour toprepare dye-bound styrene. To 5 mL of pure water deaerated by argonbubbling, 0.18 g of glycidyl methacrylate (manufactured by TokyoChemical Industry Co., Ltd.), 0.05 g of styrene (manufactured by WakoPure Chemical Industries, Ltd.), 0.05 g of divinylbenzene and 0.005 g ofthe thus obtained dye-bound styrene were added. After heating theresultant with stirring to 70° C., 0.012 g of a water-soluble azopolymerization initiator, V-50 (manufactured by Wako Pure ChemicalIndustries, Ltd.), was added and the resulting mixture was allowed toreact for 12 hours. The resulting reaction solution was centrifuged at10,000 G for 20 minutes to recover particles. The recovered particleswere purified by dispersing them in pure water and then centrifuging theresulting dispersion once again for recovery. The thus obtainedparticles were added to an excess amount of aqueous ammonia so as toconvert the epoxy groups at the particle terminals into amino groups,thereby obtaining dye-containing polystyrene nanoparticles having anamino group at a terminal.

The thus obtained dye-containing nanoparticles were adjusted with PBS(phosphate-buffered physiological saline) containing 2 mM of EDTA(ethylenediamine tetraacetic acid) to a concentration of 3 nM. Theresulting solution was mixed with SM(PEG)12 (manufactured by ThermoFisher Scientific K.K.;succinimidyl-[(N-maleimidopropionamido)-dodecaethylene glycol]ester) toa final concentration of 10 mM and allowed to react for 1 hour. The thusobtained mixture was centrifuged at 10,000 G for 20 minutes and theresulting supernatant was removed. Then, PBS containing 2 mM of EDTA wasadded to disperse the precipitates and the resulting dispersion wascentrifuged again. The precipitates were washed three times by the sameprocedure to obtain fluorescent dye-containing nanoparticles having amaleimide group at a terminal.

Meanwhile, streptavidin (manufactured by Wako Pure Chemical Industries,Ltd.) was subjected to a thiol group addition treatment withN-succinimidyl-5-acetylthioacetate (SATA) and then filtered through agel-filtration column to obtain a solution of streptavidin capable ofbinding to dye-containing nanoparticles.

The above-described fluorescent nanoparticles and streptavidin weremixed in PBS containing 2 mM of EDTA and allowed to react for 1 hour.Then, the reaction was terminated with an addition of 10 mMmercaptoethanol. After concentrating the resulting solution using acentrifugation filter, unreacted streptavidin and the like were removedusing a gel-filtration column for purification, thereby obtainingpolystyrene nanoparticles containing streptavidin-bound Texas Red dye.

(Fluorescent Label 10: Perylene Dye-Containing Polystyrene Nanoparticle)

Silica nanoparticles containing streptavidin-bound perylene dye wereobtained in the same manner as the above-described fluorescent dye 9,except that perylene diimide used in the fluorescent label 2 was used asthe fluorescent dye.

(Fluorescent Label 11: FITC Dye-Containing Polystyrene Nanoparticle)

Silica nanoparticles containing streptavidin-bound perylene dye wereobtained in the same manner as the above-described fluorescent dye 9,except that FITC used in the fluorescent dye 3 was used as thefluorescent dye.

(Fluorescent Label 12: CdSe)

Carboxylic acid-terminated CdSe/ZnS (Qdot 605, manufactured byInvitrogen) was used as fluorescent nanoparticle. The fluorescentnanoparticles were activated by EDTA (manufactured by Thermo FisherScientific K.K.) and then bound with streptavidin in the same manner asthe fluorescent label 1, thereby obtaining streptavidin-boundfluorescent nanoparticles.

(Fluorescent Label 13: CdSe-Containing Nanoparticle)

To 10 μL of CdSe/ZnS decane dispersion (Qdot 605, manufactured byInvitrogen), 0.1 mg of TEOS, 0.01 mL of ethanol and 0.03 mL ofconcentrated aqueous ammonia were added, and the resultant was stirredfor 3 hours to perform hydrolysis. The thus obtained mixture wascentrifuged at 10,000 G for 20 minutes and the resulting supernatant wasremoved. Then, ethanol was added to disperse the precipitates and theresulting dispersion was centrifuged again. The precipitates were washedtwice by the same procedure with ethanol and pure water, therebyobtaining 60 mg of CdSe-containing nanoparticles.

By SEM observation of 1,000 of the thus obtained CdSe-containingnanoparticles, the average particle size and the variation coefficientwere found to be 108 nm and 14%, respectively.

The thus obtained CdSe-containing nanoparticles were subjected tosurface amination, PEGylation and modification with streptavidin in thesame manner as in the method of preparing the fluorescent dye 3, therebyobtaining streptavidin-bound CdSe-containing nanoparticles.

The list of the fluorescent labels used is shown in Table 1.

TABLE 1 Dye or Contained Fluorescent nanoparticle particle label Type offluorescent label composition matrix Fluorescent Fluorescent dye TexasRed none label 1 Fluorescent Fluorescent dye perylene none label 2Fluorescent Fluorescent dye-containing Texas Red melamine label 3particle Fluorescent Fluorescent dye-containing perylene melamine label4 particle Fluorescent Fluorescent dye-containing fluorescein melaminelabel 5 particle Fluorescent Fluorescent dye-containing Texas Red silicalabel 6 particle Fluorescent Fluorescent dye-containing perylene silicalabel 7 particle Fluorescent Fluorescent dye-containing fluoresceinsilica label 8 particle Fluorescent Fluorescent dye-containing Texas Redpolystyrene label 9 particle Fluorescent Fluorescent dye-containingperylene polystyrene label 10 particle Fluorescent Fluorescentdye-containing fluorescein polystyrene label 11 particle FluorescentFluorescent nanoparticle CdSe none label 12 Fluorescent Fluorescent CdSesilica label 13 nanoparticle-containing particle

<Preparation of Mounting Medium>

As mounting media, the following mounting media 1-1 to 1-10, 2-1 to 2-5and 3-1 were prepared.

Discoloration Inhibitor-Containing Mounting Media Mounting Medium 1-1Rutin-Containing Mounting Medium

As a mounting medium, Entellan New (manufactured by Merck KGaA; maincomponents: acrylic resin and xylene=about 60%) or PARA Mount-N(manufactured by FALMA; main components: acrylic resin and aliphatichydrocarbons) was used as is without diluting the purchased form.

To the mounting medium, rutin was added as a discoloration inhibitor inan amount of 1% by weight (hereinafter, abbreviated as “wt %”), and theresulting mixture was stirred to obtain a discolorationinhibitor-containing mounting medium.

Mounting Medium 1-2 2,6-di-tert-butyl-4-hydroxymethylphenol-containingmounting medium

As a mounting medium, Entellan New (manufactured by Merck KGaA) or PARAMount-N (manufactured by FALMA) was used. To the mounting medium,2,6-di-tert-butyl-4-hydroxymethylphenol was added as a discolorationinhibitor in an amount of 1 wt %, and the resulting mixture was stirredto obtain a discoloration inhibitor-containing mounting medium.

Mounting Medium 1-3bis(2,2,6,6-tetramethyl-4-piperidyl)sebacate-containing mounting medium

As a mounting medium, Entellan New (manufactured by Merck KGaA) or PARAMount-N (manufactured by FALMA) was used. To the mounting medium,bis(2,2,6,6-tetramethyl-4-piperidyl)sebacate was added as adiscoloration inhibitor in an amount of 1 wt %, and the resultingmixture was stirred to obtain a discoloration inhibitor-containingmounting medium.

Mounting Medium 1-4 Phenothiazine-Containing Mounting Medium

As a mounting medium, Entellan New (manufactured by Merck KGaA) or PARAMount-N (manufactured by FALMA) was used. To the mounting medium,phenothiazine was added as a discoloration inhibitor in an amount of 1wt %, and the resulting mixture was stirred to obtain a discolorationinhibitor-containing mounting medium.

Mounting Medium 1-5 2-Mercaptobenzimidazole

As a mounting medium, Entellan New (manufactured by Merck KGaA) or PARAMount-N (manufactured by FALMA) was used. To the mounting medium,2-mercaptobenzimidazole was added as a discoloration inhibitor in anamount of 1 wt %, and the resulting mixture was stirred to obtain adiscoloration inhibitor-containing mounting medium.

Mounting Medium 1-6 Triphenyl Phosphite-Containing Mounting Medium

As a mounting medium, Entellan New (manufactured by Merck KGaA) or PARAMount-N (manufactured by FALMA) was used. To the mounting medium,triphenyl phosphite was added as a discoloration inhibitor in an amountof 1 wt %, and the resulting mixture was stirred to obtain adiscoloration inhibitor-containing mounting medium.

Mounting Medium 1-7 Dibenzyl Disulfide-Containing Mounting Medium

As a mounting medium, Entellan New (manufactured by Merck KGaA) or PARAMount-N (manufactured by FALMA) was used. To the mounting medium,dibenzyl disulfide was added as a discoloration inhibitor in an amountof 1 wt %, and the resulting mixture was stirred to obtain adiscoloration inhibitor-containing mounting medium.

Mounting Medium 1-8 didodecyl-3,3′-thiodipropionate-containing mountingmedium

As a mounting medium, Entellan New (manufactured by Merck KGaA) or PARAMount-N (manufactured by FALMA) was used. To the mounting medium,didodecyl-3,3′-thiodipropionate was added as a discoloration inhibitorin an amount of 1 wt %, and the resulting mixture was stirred to obtaina discoloration inhibitor-containing mounting medium.

Mounting Medium 1-9 DL-α-Lipoic Acid-Containing Mounting Medium

As a mounting medium, Entellan New (manufactured by Merck KGaA) or PARAMount-N (manufactured by FALMA) was used. To the mounting medium,DL-α-lipoic acid was added as a discoloration inhibitor in an amount of1 wt %, and the resulting mixture was stirred to obtain a discolorationinhibitor-containing mounting medium.

Mounting Medium 1-10 Retinoic Acid-Containing Mounting Medium

As a mounting medium, Entellan New (manufactured by Merck KGaA) or PARAMount-N (manufactured by FALMA) was used. To the mounting medium,retinoic acid was added as a discoloration inhibitor in an amount of 1wt %, and the resulting mixture was stirred to obtain a discolorationinhibitor-containing mounting medium.

Mounting Medium 2-1 Mounting Medium Containing No DiscolorationInhibitor

As a mounting medium, Entellan New (manufactured by Merck KGaA) or PARAMount-N (manufactured by FALMA) was used as is. No discolorationinhibitor was added thereto.

Mounting Medium 2-2 Cyanidin-Containing Mounting Medium

To the mounting medium, cyanidin was added as a discoloration inhibitorin an amount of 1 wt %, and the resulting mixture was stirred to obtaina discoloration inhibitor-containing mounting medium.

Mounting Medium 2-3 p-Phenylazophenol-Containing Mounting Medium

To the mounting medium, p-phenylazophenol was added as a discolorationinhibitor in an amount of 1 wt %, and the resulting mixture was stirredto obtain a discoloration inhibitor-containing mounting medium.

Mounting Medium 2-4 4-Nitroaniline-Containing Mounting Medium

As a mounting medium, Entellan New (manufactured by Merck KGaA) or PARAMount-N (manufactured by FALMA) was used. To the mounting medium,4-nitroaniline was added as a discoloration inhibitor in an amount of 1wt %, and the resulting mixture was stirred to obtain a discolorationinhibitor-containing mounting medium.

Mounting Medium 2-5 Lycopene-Containing Mounting Medium

As a mounting medium, Entellan New (manufactured by Merck KGaA) or PARAMount-N (manufactured by FALMA) was used. To the mounting medium,lycopene was added as a discoloration inhibitor in an amount of 1 wt %,and the resulting mixture was stirred to obtain a discolorationinhibitor-containing mounting medium.

Mounting Medium 3-1 DABCO-Containing Aqueous Mounting Medium

As a mounting medium, Fluoromount (manufactured by Cosmo Bio Co., Ltd.;aqueous mounting medium) was used. To this mounting medium,1,4-diazabicyclo[2.2.2]octane (DABCO) was added as a discolorationinhibitor in an amount of 1 wt %, and the resulting mixture was stirredto obtain a discoloration inhibitor-containing mounting medium.

The list of the mounting media used is shown in Table 2.

TABLE 2 Classification of discoloration inhibitor Mounting contained inmounting medium medium Discoloration inhibitor Mounting phenolicdiscoloration rutin medium1-1 inhibitor Mounting phenolic discoloration2,6-di-tert-butyl-4- medium1-2 inhibitor hydroxymethylphenol Mountingamine-based discoloration bis(2,2,6,6-tetramethyl- medium1-3 inhibitor4-piperidyl)sebacate Mounting amine-based discoloration phenothiazinemedium1-4 inhibitor Mounting phosphorus-based 2-mercaptobenzimidazolemedium1-5 discoloration inhibitor Mounting phosphorus-based triphenylphosphite medium1-6 discoloration inhibitor Mounting sulfur-baseddibenzyl disulfide medium1-7 discoloration inhibitor Mountingsulfur-based didodecyl-3,3′- medium1-8 discoloration inhibitorthiodipropionate Mounting sulfur-based DL-α-lipoic acid medium1-9discoloration inhibitor Mounting unsaturated retinoic acid medium1-10hydrocarbon-based discoloration inhibitor Mounting — none medium2-1Mounting phenolic discoloration cyanidin medium2-2 inhibitor Mountingphenolic discoloration p-phenylazophenol medium2-3 inhibitor Mountingamine-based discoloration 4-nitroaniline medium2-4 inhibitor Mountingunsaturated lycopene medium2-5 hydrocarbon-based discoloration inhibitorMounting amine-based discoloration 1,4-diazabicyclo[2.2.2]octanemedium3-1 inhibitor (DABCO)

<Tissue Staining Step> [Tissue Immunostaining]

Using the fluorescent labels 1 to 13, human breast tissue wasimmunostained. As a section to be stained, a tissue array slidemanufactured by Cosmo Bio Co., Ltd. (CB-A712) was used. After subjectingthe tissue array slide to a deparaffinization treatment, an antigenactivation treatment was performed by subjecting the tissue array slideto displacement washing with water and a 15-minute autoclave treatmentin 10 mM citrate buffer (pH 6.0). Thereafter, the tissue array slide waswashed with PBS buffer and then subjected to a 1-hour blocking treatmentwith 1% BSA-containing PBS buffer in a moist chamber. After the blockingtreatment, the tissue section was allowed to react for 2 hours withbiotinylated trastuzumab diluted with 1% BSA-containing PBS buffer to aconcentration of 0.05 nM, and the resulting tissue section wassubsequently washed. The tissue section was further allowed to reactwith the respective fluorescent labels 1 to 13 for 0.5 hour and thenwashed, thereby obtaining an immunohistochemically-stained section. Thethus obtained immunohistochemically-stained section was then immersed in4% neutral paraformaldehyde aqueous buffer for 10 minutes to perform animmobilization treatment.

[Morphological Staining]

The thus immobilized immunohistochemically-stained section was subjectedto HE staining and then immersed in ethanol for dehydration. Thedehydrated section was cleared by further immersing the section inxylene and air-drying the resulting section, thereby a double-stainedsection was obtained. It is noted here that the HE staining performed asmorphological staining does not have any effect on the below-describedevaluation of light resistance.

[Mounting]

Subsequently, the thus morphologically-stained section was mounted. Inthis process, the fluorescent labels 1 to 13 and the mounting media 1-1to 1-10, 2-1 to 2-5 and 3-1 were each used.

Evaluations of Tissue Sample Evaluation 1 Absorption by DiscolorationInhibitor

For the evaluation of absorption by a discoloration inhibitor, a xylenesolution containing the discoloration inhibitor at a concentration of 1mg/mL was prepared and placed in a 10-mm cell and the absorbance wasmeasured. When the absorbance was 0.5 or less at both 450 nm and 600 nm,the absorption by the discoloration inhibitor was evaluated as “absent”and, when the absorbance was higher than 0.5 at either or both of thesewavelengths, the absorption by the discoloration inhibitor was evaluatedas “present”. For the measurement of the absorbance, aspectrophotometer, U-4100 manufactured by Hitachi, Ltd., was used. It isnoted here that, when the solubility of the subject discolorationinhibitor in xylene was poor, a 0.5-wt % xylene solution of thediscoloration inhibitor was prepared and placed in a 10-mm cell tocarryout the measurement. When the solubility in xylene was even worse,the solvent was changed to a mixed solvent of xylene and EtOH or thelike to carry out the measurement.

Evaluation 2 Evaluation of Light Resistance Before and after 30-MinuteIrradiation, and Improvement Rate

The tissue sections that were immunostained with the respectivefluorescent labels 1 to 13 and then mounted with the respective mountingmedia 1-1 to 1-10 and 2-1 to 2-5 were each allowed to emit fluorescenceby irradiation with excitation light. For each of the resulting tissuesections, an image was obtained using an upright-type fluorescencemicroscope (manufactured by Carl Zeiss; Axio Imager 2 equipped with amonochrome camera, AxioCam MRm).

The excitation wavelength (nm) and the fluorescence wavelength (nm) wereset using an optical filter. For Texas Red, perylene imide and thosenanoparticles containing Texas Red or perylene imide, the excitationwavelength and the fluorescence wavelength were set to be 575 to 600 nmand 612 to 682 nm, respectively. For fluorescein and thefluorescein-containing nanoparticles, the excitation wavelength and thefluorescence wavelength were set to be 450 to 490 nm and 500 to 550 nm,respectively. Further, for CdSe and the CdSe-containing nanoparticles,the excitation wavelength and the fluorescence wavelength were set to be345 to 395 nm and 600 to 700 nm, respectively.

The conditions of the excitation wavelength in the microscopeobservation and image acquisition were set such that the intensity ofthe irradiation light in the vicinity of the center of the visual fieldwas 900 W/cm² for excitation at 575 to 600 nm and 450 to 490 nm, and 500W/cm² for excitation at 365 nm and 345 to 395 nm. Here, the intensity ofthe irradiation light was determined by measuring the light energy valueusing a power meter equipped with a×40 objective lens, 8230 manufacturedby Advantest Corporation, and then dividing the measured value by theirradiated visual area of the ×40 objective lens (about φ50 μm). In theimage acquisition process, the exposure time was arbitrarily set suchthat the image brightness was not saturated. The measurement wasperformed at an exposure time of, for example, 1,000 ms. It is notedhere that the acquired images were not corrected and the brightnessvalue was adjusted to be linear over the entire range.

The brightness of each pixel was calculated from the thus obtainedrespective images using an image analysis software, Image-J(manufactured by the U.S. National Institutes of Health), and theaverage brightness of the sites that were stained with each fluorescentlabel (immunostained parts) was calculated (brightness of theimmunostained parts). This average brightness corresponds to a signalvalue (S). In the 256-gradation data, a brightness of “0” is defined asblack (the darkest) and a brightness of “255” is defined as white (thebrightest). When an image having more than 256 color gradations wasused, the calculated brightness value was divided by the maximumgradation value and then multiplied by 255 so that the brightness can becompared in terms of 256-gradation scale.

The light resistance of each fluorescent label was evaluated bycontinuing the irradiation of excitation light with the visual fieldbeing fixed for 30 minutes, determining the brightness of theimmunostained parts from both of the micrographs that were takenimmediately after the start of the irradiation (at the start of theirradiation, 0 minute) and after the irradiation (30 minutes), and thencalculating the brightness retention rate (%), which is represented byan equation: (brightness after 30-minute irradiation)/(brightnessimmediately after the start of irradiation)×100.

Further, for comparison of the effects of the discoloration inhibitors,the same operations were performed using the corresponding mountingmedia containing no discoloration inhibitor and the improvement rate,which is represented by an equation: (brightness retention rate with thediscoloration inhibitor)/(brightness retention rate without thediscoloration inhibitor)×100, was calculated.

Evaluation 3 Storage Property

For each of the mounted section slides, the brightness after 3-monthstorage was evaluated. The evaluation of the brightness was performed inthe same manner as Evaluation 2. The storage property was evaluated as“◯” (present) when 70% or more of the initial brightness was retained,and it was evaluated as “x” (absent) when the brightness was reduced toless than 70% of the initial brightness.

Evaluation 4 Transparency

For each of the mounted section slides, using a spectrophotometer U-4100(manufactured by Hitachi, Ltd.), the absorbance at 450 to 600 nm wasmeasured and evaluated. An evaluation of “◯” (transparent) was givenwhen the absorbance was 0.1 or less at both 450 nm and 600 nm, and anevaluation of “x” (non-transparent) was given when the absorbance washigher than 0.1 at either or both of these wavelengths. When themounting medium is colored, the transparency is reduced. In addition,when the tissue section develops cloudiness, the transparency is alsoreduced. Thus, from the standpoint of maintaining the brightness of thefluorescent label, it is preferred that the section slide betransparent.

Evaluation 5 Stain Retainability

In order to confirm that each tissue section was stained with therespective oil-based mounting media, a sample of each tissue section notsubjected to the immobilization step at the time of staining wasprepared and compared with the section sample that was subjected to theimmobilization step. Comparing against the sample not subjected to theimmobilization step, when the initial brightness was increased to1.1-fold or higher, the fluorescent staining properties were evaluatedas “◯” (maintained) and, when the initial brightness was reduced to lessthan 1.1-fold, the fluorescent staining properties were evaluated as “x”(not maintained).

The results of the above-described evaluations are shown in Tables 3-1to 3-7, 4, 5-1 to 5-3 and 6.

TABLE 3-1 Maintenance Absorption of Mount- by Brightness Improve-fluorescent ing Fluorescent Immobilization Discoloration discolorationretention ment staining Storage Trans- medium label treatment inhibitorinhibitor rate [%] rate [%] properties property parency Example 1 En 1done rutin absent 58 383 ∘ ∘ ∘ Example 2 En 1 done 2,6-di-tert-butyl-4-absent 53 355 ∘ ∘ ∘ hydroxymethylphenol Example 3 En 1 donebis(2,2,6,6-tetramethyl- absent 52 344 ∘ ∘ ∘ 4-piperidyl)sebacateExample 4 En 1 done phenothiazine absent 49 327 ∘ ∘ ∘ Example 5 En 1done 2-mercaptobenzimidazole absent 43 284 ∘ ∘ ∘ Example 6 En 1 donetriphenyl phosphite absent 44 291 ∘ ∘ ∘ Example 7 En 1 done dibenzyldisulfide absent 41 270 ∘ ∘ ∘ Example 8 En 1 done didodecyl-3,3′- absent42 277 ∘ ∘ ∘ thiodipropionate Example 9 En 1 done DL-α-lipoic acidabsent 39 263 ∘ ∘ ∘ Example En 1 done retinoic acid absent 41 270 ∘ ∘ ∘10 Example En 2 done rutin absent 65 217 ∘ ∘ ∘ 11 Example En 2 done2,6-di-tert-butyl-4- absent 62 205 ∘ ∘ ∘ 12 hydroxymethylphenol ExampleEn 2 done bis(2,2,6,6-tetramethyl- absent 60 200 ∘ ∘ ∘ 134-piperidyl)sebacate Example En 2 done phenothiazine absent 58 193 ∘ ∘ ∘14 Example En 2 done 2-mercaptobenzimidazole absent 53 176 ∘ ∘ ∘ 15Example En 2 done triphenyl phosphite absent 54 179 ∘ ∘ ∘ 16 Example En2 done dibenzyl disulfide absent 51 170 ∘ ∘ ∘ 17 Example En 2 donedidodecyl-3,3′- absent 52 173 ∘ ∘ ∘ 18 thiodipropionate Example En 2done DL-α-lipoic acid absent 50 167 ∘ ∘ ∘ 19 Example En 2 done retinoicacid absent 51 170 ∘ ∘ ∘ 20 En: Entellan New

TABLE 3-2 Maintenance Absorption of Mount- by Brightness Improve-fluorescent ing Fluorescent Immobilization Discoloration discolorationretention ment staining Storage Trans- medium label treatment inhibitorinhibitor rate [%] rate [%] properties property parency Example En 3done rutin absent 65 217 ∘ ∘ ∘ 21 Example En 3 done 2,6-di-tert-butyl-4-absent 62 205 ∘ ∘ ∘ 22 hydroxymethylphenol Example En 3 donebis(2,2,6,6-tetramethyl- absent 60 200 ∘ ∘ ∘ 23 4-piperidyl)sebacateExample En 3 done phenothiazine absent 58 193 ∘ ∘ ∘ 24 Example En 3 done2-mercaptobenzimidazole absent 53 176 ∘ ∘ ∘ 25 Example En 3 donetriphenyl phosphite absent 54 179 ∘ ∘ ∘ 26 Example En 3 done dibenzyldisulfide absent 51 170 ∘ ∘ ∘ 27 Example En 3 done didodecyl-3,3′-absent 52 173 ∘ ∘ ∘ 28 thiodipropionate Example En 3 done DL-α-lipoicacid absent 50 167 ∘ ∘ ∘ 29 Example En 3 done retinoic acid absent 51170 ∘ ∘ ∘ 30 Example En 4 done rutin absent 80 133 ∘ ∘ ∘ 31 Example En 4done 2,6-di-tert-butyl-4- absent 78 130 ∘ ∘ ∘ 32 hydroxymethylphenolExample En 4 done bis(2,2,6,6-tetramethyl- absent 77 129 ∘ ∘ ∘ 334-piperidyl)sebacate Example En 4 done phenothiazine absent 76 127 ∘ ∘ ∘34 Example En 4 done 2-mercaptobenzimidazole absent 73 122 ∘ ∘ ∘ 35Example En 4 done triphenyl phosphite absent 74 123 ∘ ∘ ∘ 36 Example En4 done dibenzyl disulfide absent 72 120 ∘ ∘ ∘ 37 Example En 4 donedidodecyl-3,3′- absent 73 121 ∘ ∘ ∘ 38 thiodipropionate Example En 4done DL-α-lipoic acid absent 72 119 ∘ ∘ ∘ 39 Example En 4 done retinoicacid absent 72 120 ∘ ∘ ∘ 40 En: Entellan New

TABLE 3-3 Maintenance Absorption of Mount- by Brightness Improve-fluorescent ing Fluorescent Immobilization Discoloration discolorationretention ment staining Storage Trans- medium label treatment inhibitorinhibitor rate [%] rate [%] properties property parency Example En 5done rutin absent 65 217 ∘ ∘ ∘ 41 Example En 5 done 2,6-di-tert-butyl-4-absent 62 205 ∘ ∘ ∘ 42 hydroxymethylphenol Example En 5 donebis(2,2,6,6-tetramethyl- absent 60 200 ∘ ∘ ∘ 43 4-piperidyl)sebacateExample En 5 done phenothiazine absent 58 193 ∘ ∘ ∘ 44 Example En 5 done2-mercaptobenzimidazole absent 53 176 ∘ ∘ ∘ 45 Example En 5 donetriphenyl phosphite absent 54 179 ∘ ∘ ∘ 46 Example En 5 done dibenzyldisulfide absent 51 170 ∘ ∘ ∘ 47 Example En 5 done didodecyl-3,3′-absent 52 173 ∘ ∘ ∘ 48 thiodipropionate Example En 5 done DL-α-lipoicacid absent 50 167 ∘ ∘ ∘ 49 Example En 5 done retinoic acid absent 51170 ∘ ∘ ∘ 50 Example En 6 done rutin absent 63 250 ∘ ∘ ∘ 51 Example En 6done 2,6-di-tert-butyl-4- absent 59 235 ∘ ∘ ∘ 52 hydroxymethylphenolExample En 6 done bis(2,2,6,6-tetramethyl- absent 57 229 ∘ ∘ ∘ 534-piperidyl)sebacate Example En 6 done phenothiazine absent 55 220 ∘ ∘ ∘54 Example En 6 done 2-mercaptobenzimidazole absent 49 198 ∘ ∘ ∘ 55Example En 6 done triphenyl phosphite absent 50 201 ∘ ∘ ∘ 56 Example En6 done dibenzyl disulfide absent 48 190 ∘ ∘ ∘ 57 Example En 6 donedidodecyl-3,3′- absent 48 194 ∘ ∘ ∘ 58 thiodipropionate Example En 6done DL-α-lipoic acid absent 47 186 ∘ ∘ ∘ 59 Example En 6 done retinoicacid absent 48 190 ∘ ∘ ∘ 60 En: Entellan New

TABLE 3-4 Maintenance Absorption of Mount- by Brightness Improve-fluorescent ing Fluorescent Immobilization Discoloration discolorationretention ment staining Storage Trans- medium label treatment inhibitorinhibitor rate [%] rate [%] properties property parency Example En 7done rutin absent 78 141 ∘ ∘ ∘ 61 Example En 7 done 2,6-di-tert-butyl-4-absent 75 137 ∘ ∘ ∘ 62 hydroxymethylphenol Example En 7 donebis(2,2,6,6-tetramethyl- absent 74 135 ∘ ∘ ∘ 63 4-piperidyl)sebacateExample En 7 done phenothiazine absent 73 133 ∘ ∘ ∘ 64 Example En 7 done2-mercaptobenzimidazole absent 70 127 ∘ ∘ ∘ 65 Example En 7 donetriphenyl phosphite absent 70 128 ∘ ∘ ∘ 66 Example En 7 done dibenzyldisulfide absent 69 125 ∘ ∘ ∘ 67 Example En 7 done didodecyl-3,3′-absent 69 126 ∘ ∘ ∘ 68 thiodipropionate Example En 7 done DL-α-lipoicacid absent 68 124 ∘ ∘ ∘ 69 Example En 7 done retinoic acid absent 69125 ∘ ∘ ∘ 70 Example En 8 done rutin absent 63 250 ∘ ∘ ∘ 71 Example En 8done 2,6-di-tert-butyl-4- absent 59 235 ∘ ∘ ∘ 72 hydroxymethylphenolExample En 8 done bis(2,2,6,6-tetramethyl- absent 57 229 ∘ ∘ ∘ 734-piperidyl)sebacate Example En 8 done phenothiazine absent 55 220 ∘ ∘ ∘74 Example En 8 done 2-mercaptobenzimidazole absent 49 198 ∘ ∘ ∘ 75Example En 8 done triphenyl phosphite absent 50 201 ∘ ∘ ∘ 76 Example En8 done dibenzyl disulfide absent 48 190 ∘ ∘ ∘ 77 Example En 8 donedidodecyl-3,3′- absent 48 194 ∘ ∘ ∘ 78 thiodipropionate Example En 8done DL-α-lipoic acid absent 47 186 ∘ ∘ ∘ 79 Example En 8 done retinoicacid absent 48 190 ∘ ∘ ∘ 80 En: Entellan New

TABLE 3-5 Maintenance Absorption of Mount- by Brightness Improve-fluorescent ing Fluorescent Immobilization Discoloration discolorationretention ment staining Storage Trans- medium label treatment inhibitorinhibitor rate [%] rate [%] properties property parency Example En 9done rutin absent 70 175 ∘ ∘ ∘ 81 Example En 9 done 2,6-di-tert-butyl-4-absent 67 168 ∘ ∘ ∘ 82 hydroxymethylphenol Example En 9 donebis(2,2,6,6-tetramethyl- absent 66 165 ∘ ∘ ∘ 83 4-piperidyl)sebacateExample En 9 done phenothiazine absent 64 160 ∘ ∘ ∘ 84 Example En 9 done2-mercaptobenzimidazole absent 60 149 ∘ ∘ ∘ 85 Example En 9 donetriphenyl phosphite absent 60 151 ∘ ∘ ∘ 86 Example En 9 done dibenzyldisulfide absent 58 145 ∘ ∘ ∘ 87 Example En 9 done didodecyl-3,3′-absent 59 147 ∘ ∘ ∘ 88 thiodipropionate Example En 9 done DL-α-lipoicacid absent 57 143 ∘ ∘ ∘ 89 Example En 9 done retinoic acid absent 58145 ∘ ∘ ∘ 90 Example En 10 done rutin absent 85 121 ∘ ∘ ∘ 91 Example En10 done 2,6-di-tert-butyl-4- absent 84 119 ∘ ∘ ∘ 92 hydroxymethylphenolExample En 10 done bis(2,2,6,6-tetramethyl- absent 83 118 ∘ ∘ ∘ 934-piperidyl)sebacate Example En 10 done phenothiazine absent 82 117 ∘ ∘∘ 94 Example En 10 done 2-mercaptobenzimidazole absent 80 114 ∘ ∘ ∘ 95Example En 10 done triphenyl phosphite absent 80 114 ∘ ∘ ∘ 96 Example En10 done dibenzyl disulfide absent 79 113 ∘ ∘ ∘ 97 Example En 10 donedidodecyl-3,3′- absent 79 113 ∘ ∘ ∘ 98 thiodipropionate Example En 10done DL-α-lipoic acid absent 79 112 ∘ ∘ ∘ 99 Example En 10 done retinoicacid absent 79 113 ∘ ∘ ∘ 100 En: Entellan New

TABLE 3-6 Maintenance Absorption of Mount- by Brightness Improve-fluorescent ing Fluorescent Immobilization Discoloration discolorationretention ment staining Storage Trans- medium label treatment inhibitorinhibitor rate [%] rate [%] properties property parency Example En 11done rutin absent 70 175 ∘ ∘ ∘ 101 Example En 11 done2,6-di-tert-butyl-4- absent 67 168 ∘ ∘ ∘ 102 hydroxymethylphenol ExampleEn 11 done bis(2,2,6,6-tetramethyl- absent 66 165 ∘ ∘ ∘ 1034-piperidyl)sebacate Example En 11 done phenothiazine absent 64 160 ∘ ∘∘ 104 Example En 11 done 2-mercaptobenzimidazole absent 60 149 ∘ ∘ ∘ 105Example En 11 done triphenyl phosphite absent 60 151 ∘ ∘ ∘ 106 ExampleEn 11 done dibenzyl disulfide absent 58 145 ∘ ∘ ∘ 107 Example En 11 donedidodecyl-3,3′- absent 59 147 ∘ ∘ ∘ 108 thiodipropionate Example En 11done DL-α-lipoic acid absent 57 143 ∘ ∘ ∘ 109 Example En 11 doneretinoic acid absent 58 145 ∘ ∘ ∘ 110 Example En 12 done rutin absent 88117 ∘ ∘ ∘ 111 Example En 12 done 2,6-di-tert-butyl-4- absent 86 115 ∘ ∘∘ 112 hydroxymethylphenol Example En 12 done bis(2,2,6,6-tetramethyl-absent 86 114 ∘ ∘ ∘ 113 4-piperidyl)sebacate Example En 12 donephenothiazine absent 85 113 ∘ ∘ ∘ 114 Example En 12 done2-mercaptobenzimidazole absent 83 111 ∘ ∘ ∘ 115 Example En 12 donetriphenyl phosphite absent 83 111 ∘ ∘ ∘ 116 Example En 12 done dibenzyldisulfide absent 83 110 ∘ ∘ ∘ 117 Example En 12 done didodecyl-3,3′-absent 83 110 ∘ ∘ ∘ 118 thiodipropionate Example En 12 done DL-α-lipoicacid absent 82 110 ∘ ∘ ∘ 119 Example En 12 done retinoic acid absent 90105 ∘ ∘ ∘ 120 En: Entellan New

TABLE 3-7 Maintenance Absorption of Mount- by Brightness Improve-fluorescent ing Fluorescent Immobilization Discoloration discolorationretention ment staining Storage Trans- medium label treatment inhibitorinhibitor rate [%] rate [%] properties property parency Example En 13done rutin absent 93 109 ∘ ∘ ∘ 121 Example En 13 done2,6-di-tert-butyl-4- absent 92 108 ∘ ∘ ∘ 122 hydroxymethylphenol ExampleEn 13 done bis(2,2,6,6-tetramethyl- absent 91 108 ∘ ∘ ∘ 1234-piperidyl)sebacate Example En 13 done phenothiazine absent 91 107 ∘ ∘∘ 124 Example En 13 done 2-mereaptobenzimidazole absent 90 106 ∘ ∘ ∘ 125Example En 13 done triphenyl phosphite absent 90 106 ∘ ∘ ∘ 126 ExampleEn 13 done dibenzyl disulfide absent 90 105 ∘ ∘ ∘ 127 Example En 13 donedidodecyl-3,3′- absent 90 106 ∘ ∘ ∘ 128 thiodipropionate Example En 13done DL-α-lipoic acid absent 89 105 ∘ ∘ ∘ 129 Example En 13 doneretinoic acid absent 90 105 ∘ ∘ ∘ 130 Example Pm 3 done rutin absent 65217 ∘ ∘ ∘ 131 Example Pm 3 done 2,6-di-tert-butyl-4- absent 62 205 ∘ ∘ ∘132 hydroxymethylphenol Example Pm 3 done bis(2,2,6,6-tetramethyl-absent 60 200 ∘ ∘ ∘ 133 4-piperidyl)sebacate Example Pm 3 donephenothiazine absent 58 193 ∘ ∘ ∘ 134 Example Pm 3 done2-mercaptobenzimidazole absent 53 176 ∘ ∘ ∘ 135 Example Pm 3 donetriphenyl phosphite absent 54 179 ∘ ∘ ∘ 136 Example Pm 3 done dibenzyldisulfide absent 51 170 ∘ ∘ ∘ 137 Example Pm 3 done didodecyl-3,3′-absent 52 173 ∘ ∘ ∘ 138 thiodipropionate Example Pm 3 done DL-α-lipoicacid absent 50 167 ∘ ∘ ∘ 139 Example Pm 3 done retinoic acid absent 51170 ∘ ∘ ∘ 140

TABLE 4 Maintenance of Absorption by Brightness Improve- fluorescentMounting Fluorescent Immobilization Discoloration discolorationretention ment staining Storage Trans- medium label treatment inhibitorinhibitor rate [%] rate [%] properties property parency Example 141 En 1done none absent 15 — ∘ ∘ ∘ Example 142 En 2 done none absent 30 — ∘ ∘ ∘Example 143 En 3 done none absent 30 — ∘ ∘ ∘ Example 144 En 4 done noneabsent 60 — ∘ ∘ ∘ Example 145 En 5 done none absent 30 — ∘ ∘ ∘ Example146 En 6 done none absent 25 — ∘ ∘ ∘ Example 147 En 7 done none absent55 — ∘ ∘ ∘ Example 148 En 8 done none absent 25 — ∘ ∘ ∘ Example 149 En 9done none absent 40 — ∘ ∘ ∘ Example 150 En 10 done none absent 70 — ∘ ∘∘ Example 151 En 11 done none absent 40 — ∘ ∘ ∘ Example 152 En 12 donenone absent 75 — ∘ ∘ ∘ Example 153 En 13 done none absent 85 — ∘ ∘ ∘Example 154 Pm 3 done none absent 30 — ∘ ∘ ∘ En: Entellan New, Pm: PARAMount-N

TABLE 5-1 Maintenance of Absorption by Brightness Improve- fluorescentMounting Fluorescent Immobilization Discoloration discolorationretention ment staining Storage Trans- medium label treatment inhibitorinhibitor rate [%] rate [%] properties property parency Example En 1done cyanidin absent 58 383 ∘ ∘ x 155 Example En 1 donep-phenylazophenol absent 49 327 ∘ ∘ x 156 Example En 1 done4-nitroaniline absent 47 315 ∘ ∘ x 157 Example En 1 done lycopene absent42 281 ∘ ∘ x 158 Example En 2 done cyanidin absent 65 217 ∘ ∘ x 159Example En 2 done p-phenylazophenol absent 58 193 ∘ ∘ x 160 Example En 2done 4-nitroaniline absent 57 189 ∘ ∘ x 161 Example En 2 done lycopeneabsent 52 175 ∘ ∘ x 162 Example En 3 done cyanidin absent 65 217 ∘ ∘ x163 Example En 3 done p-phenylazophenol absent 58 193 ∘ ∘ x 164 ExampleEn 3 done 4-nitroaniline absent 57 189 ∘ ∘ x 165 Example En 3 donelycopene absent 52 175 ∘ ∘ x 166 Example En 4 done cyanidin absent 80133 ∘ ∘ x 167 Example En 4 done p-phenylazophenol absent 76 127 ∘ ∘ x168 Example En 4 done 4-nitroaniline absent 75 125 ∘ ∘ x 169 Example En4 done lycopene absent 73 121 ∘ ∘ x 170 Example En 5 done cyanidinabsent 65 217 ∘ ∘ x 171 Example En 5 done p-phenylazophenol absent 58193 ∘ ∘ x 172 Example En 5 done 4-nitroaniline absent 57 189 ∘ ∘ x 173Example En 5 done lycopene absent 52 175 ∘ ∘ x 174 En: Entellan New

TABLE 5-2 Maintenance of Absorption by Brightness Improve- fluorescentMounting Fluorescent Immobilization Discoloration discolorationretention ment staining Storage Trans- medium label treatment inhibitorinhibitor rate [%] rate [%] properties property parency Example En 6done cyanidin absent 63 250 ∘ ∘ x 175 Example En 6 donep-phenylazophenol absent 55 220 ∘ ∘ x 176 Example En 6 done4-nitroaniline absent 54 214 ∘ ∘ x 177 Example En 6 done lycopene absent49 196 ∘ ∘ x 178 Example En 7 done cyanidin absent 78 141 ∘ ∘ x 179Example En 7 done p-phenylazophenol absent 73 133 ∘ ∘ x 180 Example En 7done 4-nitroaniline absent 72 131 ∘ ∘ x 181 Example En 7 done lycopeneabsent 69 126 ∘ ∘ x 182 Example En 8 done cyanidin absent 63 250 ∘ ∘ x183 Example En 8 done p-phenylazophenol absent 55 220 ∘ ∘ x 184 ExampleEn 8 done 4-nitroaniline absent 54 214 ∘ ∘ x 185 Example En 8 donelycopene absent 49 196 ∘ ∘ x 186 Example En 9 done cyanidin absent 70175 ∘ ∘ x 187 Example En 9 done p-phenylazophenol absent 64 160 ∘ ∘ x188 Example En 9 done 4-nitroaniline absent 63 157 ∘ ∘ x 189 Example En9 done lycopene absent 59 148 ∘ ∘ x 190 Example En 10 done cyanidinabsent 85 121 ∘ ∘ x 191 Example En 10 done p-phenylazophenol absent 82117 ∘ ∘ x 192 Example En 10 done 4-nitroaniline absent 81 116 ∘ ∘ x 193Example En 10 done lycopene absent 80 114 ∘ ∘ x 194 En: Entellan New

TABLE 5-3 Maintenance of Absorption by Brightness Improve- fluorescentMounting Fluorescent Immobilization Discoloration discolorationretention ment staining Storage Trans- medium label treatment inhibitorinhibitor rate [%] rate [%] properties property parency Example En 11done cyanidin absent 70 175 ∘ ∘ x 195 Example En 11 donep-phenylazophenol absent 64 160 ∘ ∘ x 196 Example En 11 done4-nitroaniline absent 63 157 ∘ ∘ x 197 Example En 11 done lycopeneabsent 59 148 ∘ ∘ x 198 Example En 12 done cyanidin absent 88 117 ∘ ∘ x199 Example En 12 done p-phenylazophenol absent 85 113 ∘ ∘ x 200 ExampleEn 12 done 4-nitroaniline absent 85 113 ∘ ∘ x 201 Example En 12 donelycopene absent 90 106 ∘ ∘ x 202 Example En 13 done cyanidin absent 93109 ∘ ∘ x 203 Example En 13 done p-phenylazophenol absent 91 107 ∘ ∘ x204 Example En 13 done 4-nitroaniline absent 91 107 ∘ ∘ x 205 Example En13 done lycopene absent 90 106 ∘ ∘ x 206 En: Entellan New

TABLE 6 Maintenance of Absorption by Brightness Improve- fluorescentMounting Fluorescent Immobilization Discoloration discolorationretention ment staining Storage Trans- medium label treatment inhibitorinhibitor rate [%] rate [%] properties property parency Comparative Fm 3done absent absent 15 — ∘ x x Example 1 Comparative En 1 absent absentabsent n.d. — x ∘ ∘ Example 2 Comparative En 2 absent absent absent n.d.— x ∘ ∘ Example 3 Comparative En 3 absent absent absent n.d. — x ∘ ∘Example 4 Comparative En 4 absent absent absent n.d. — x ∘ ∘ Example 5Comparative En 5 absent absent absent n.d. — x ∘ ∘ Example 6 ComparativeEn 6 absent absent absent n.d. — x ∘ ∘ Example 7 Comparative En 7 absentabsent absent n.d. — x ∘ ∘ Example 8 Comparative En 8 absent absentabsent n.d. — x ∘ ∘ Example 9 Comparative En 9 absent absent absent n.d.— x ∘ ∘ Example 10 Comparative En 10 absent absent absent n.d. — x ∘ ∘Example 11 Comparative En 11 absent absent absent n.d. — x ∘ ∘ Example12 Comparative En 12 absent absent absent n.d. — x ∘ ∘ Example 13Comparative En 13 absent absent absent n.d. — x ∘ ∘ Example 14Comparative Pm 3 absent absent absent n.d. — x ∘ ∘ Example 15 En:Entellan New, Pm: PARA Mount-N, Fm: Fluoromount n.d.: not determined

As a result of these evaluations, it was shown that, even when amounting medium comprising a solvent not freely miscible with water(oil-based mounting medium) is used, by performing an immobilizationtreatment, a fluorescently stained specimen which has no reduction inthe fluorescent staining properties (reduction of the light-emittingparts) and no bleeding of stains and may thus be actually used forobservation can be prepared. In addition, it was shown that an additionof a discoloration inhibitor improves the brightness retention rate.Furthermore, it was also shown that, by using a discoloration inhibitorshowing no absorption, a transparent section slide that is more suitablefor fluorescence observation can be obtained.

1. A biological substance detection method for specifically detecting abiological substance from a pathological specimen, said methodcomprising the steps of: immunostaining said specimen with a fluorescentlabel; immobilizing the thus stained specimen; and mounting the thusimmobilized specimen using a mounting medium comprising an organicsolvent not freely miscible with water.
 2. The biological substancedetection method according to claim 1, wherein said fluorescent labelcomprises at least one selected from the group consisting of fluorescentdye-containing nanoparticles and fluorescent nanoparticle-containingparticles.
 3. The biological substance detection method according toclaim 1, wherein said organic solvent not freely miscible with watercomprises at least one selected from the group consisting of aromatichydrocarbons, unsaturated hydrocarbons, ketones, esters, ethers andalcohols.
 4. The biological substance detection method according toclaim 1, wherein said organic solvent not freely miscible with watercomprises at least one selected from the group consisting of xylene,toluene and limonene.
 5. The biological substance detection methodaccording to claim 1, wherein said mounting medium comprises adiscoloration inhibitor.
 6. The biological substance detection methodaccording to claim 5, wherein said discoloration inhibitor does notabsorb light in an absorption wavelength range of 450 to 600 nm.
 7. Thebiological substance detection method according to claim 5, wherein asection slide prepared using said mounting medium comprising saiddiscoloration inhibitor is transparent.
 8. The biological substancedetection method according to claim 5, wherein said discolorationinhibitor comprises at least one selected from the group consisting ofphenols, amines, thiols, sulfides, disulfides and unsaturatedhydrocarbons.
 9. The biological substance detection method according toclaim 5, wherein said discoloration inhibitor comprises at least oneselected from the group consisting of phenols and amines.
 10. Abiological substance detection kit used for the biological substancedetection method according to claim 1, said kit comprising: a mountingmedium comprising an organic solvent not freely miscible with water; andan instruction manual that describes said biological substance detectionmethod.
 11. A biological substance detection kit used for the biologicalsubstance detection method according to claim 5, said kit comprising: amounting medium comprising an organic solvent not freely miscible withwater and a discoloration inhibitor; and an instruction manual thatdescribes said biological substance detection method.
 12. The biologicalsubstance detection kit according to claim 10, which further comprises afluorescent label for immunostaining and a fluorescent labeling reagent.13. A pathological specimen used for the biological substance detectionmethod according to claim 1, wherein said pathological specimen issubjected to: an immunostaining treatment using a fluorescent label forspecifically detecting a biological substance; an immobilizationtreatment; and a mounting treatment using a mounting medium comprisingan organic solvent not freely miscible with water.